Pt(IV) antitumor agent

ABSTRACT

An improved platinum(IV) compound and its application as an antitumor agent are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/547,074filed Apr. 11, 2000, Provisional No. 60,128,939 Apr. 13, 1999 thecontents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to the area of platinum antitumor drugs.In particular, it relates to a specific platinum(IV) complex, and itsapplication as an antitumor agent in the treatment of cancer.

BACKGROUND ART

Interest in platinum-based antitumor drugs has its origin in thediscovery of the inhibitory effects of platinum complexes on celldivision. Subsequent research and development led to the approval of theuse of cisplatin, [PtCl₂(NH₃)₂] for cancer therapy. Cisplatin is nowwidely accepted and is one of the three most widely utilized antitumordrugs in the world. However, cisplatin has several disadvantages thatinclude severe toxicity such as nephrotoxicity, neurotoxicity andemetogenesis. Cisplatin also has limited aqueous solubility (1 mg/ml)and is effective in a narrow range of tumors. Some tumors have naturalresistance to platinum drugs while others develop resistance after theinitial treatment.

In addition to cisplatin, carboplatin ordiammine[1,1-cyclobutanedicarboxylato (2-)]-O,O′-platinum(II) has alsoreceived worldwide approval for use in cancer therapy. Carboplatin isless toxic than cisplatin and has greater aqueous solubility (14 mg/ml)but it is still only active in the same range of tumors as cisplatin.Many platinum complexes have been studied in an attempt to overcome thelimitation of cisplatin (Wong, E.; et al., Chem. Rev. 1999, 9,2451-2466).

A class of platinum compounds that has been reported to have activityagainst cancer is mixed amine platinum complexes of the general formulaPt(L)(L′)A₂ or Pt(L)(L′)A₂B₂ where L and L′ are different amines, andwhere one of the amines sterically hinders access of Pt to the DNA ofthe tumor cell. One example within this class of compounds iscis-ammine(2-methylpyridine)dichloroplatinum(II) (Holford, J. F.; etal., Anti-Cancer Drug Des. 1998, 13, 1 and Raynaud, F. I., et al., Clin.Cancer Res. 1997, 3, 206-2074 and Holford, J. F., et al., Br. J Cancer1999 77, 366 and U.S. Pat. No. 5,665,771). Other examples of antitumorcompounds are bis-acetatoammine(cyclohexylamine)dichloroplatinum(IV) andbis-butyratoammine(cyclohexylamine)dichloroplatinum(IV) (Kelland, L. R.;et al., Cancer Res. 1992, 52, 3857; Kelland, L. R., et al., Cancer Res.1992, 52, 822; Kelland, L. R.; et al., Cancer Res. 1993, 53, 2581 andU.S. Pat. No. 5,244,919.

The mixed amine platinum compounds have been reported to have antitumoractivity in cisplatin resistant tumors. To compare the ability ofvarious compounds to overcome platinum drug resistance, resistancefactors are calculated for sets of cell lines. The resistance factor isdefined as the ratio:$\frac{{activity}\quad {against}\quad a\quad {parent}\quad {line}\quad {of}\quad {cancer}\quad {cells}}{\begin{matrix}{{avtivity}\quad {against}\quad a\quad {derivative}\quad {of}\quad {that}\quad {cell}\quad {line}} \\{{that}\quad {has}\quad {developed}\quad {resistance}\quad {to}\quad {cisplatin}}\end{matrix}}$

Thus, small resistance factors are preferred since the compound isbetter able to overcome the drug resistance of the cancer cells. Theabove cited compounds, Cis-ammine(2-methylpyridine)dichloroplatinum(II),bis-acetatoammine(cyclohexylamine)dichloroplatinum(IV) andbis-butyratoammine-(cyclohexylamine)dichloroplatinum(IV) were reportedto have activity in certain cisplatin resistant tumors, but do not haveequivalent activity in all cancer cells with different mechanisms ofplatinum drug resistance. For example,bis-acetatoammine(cyclohexylamine)dichloroplatinum(IV) andbis-butyratoammine(cyclohexyl-amine)dichloroplatinum(IV) have lowerresistance factors in the set of 41M/41MR cancer cell lines than in theA2780/A2780R or CH1/CH1R sets of cells. On the other hand,cis-ammine(2-methylpyridine)dichloroplatinum(II) has a higher resistancefactor in A2780/A2780R cell lines than in the 41M/41MR and CH1/CH1R celllines. The platinum drug resistance in 41MR cells is due to reducedplatinum accumulation, while in CH1R cells, the resistance is due toenhanced removal of and/or increased tolerance to Pt-DNA adducts.Resistance in A2780R cancer cells is due to detoxification via elevatedglutathione levels, decreased uptake and increased DNA repair.

Despite the improved activity of these mixed amine platinum compoundscompared to cisplatin in some platinum drug resistant cancer cells,their solubility in aqueous solution is limited; their aqueoussolubility at ambient temperature and neutral pH are even lower thanthat of cisplatin. The limited aqueous solubility poses difficulties inthe formulation and administration of these compounds. In particular,the iv administration of a platinum drug with low aqueous solubility mayrequire the infusion of a large volume of liquid in order to achieve thetherapeutic dose; a potentially long and inconvenient process.

Increasing the water solubility of platinum antitumor compounds has beenan important practical objective of many platinum drug developmentprograms. The solubility of cisplatin (˜1 mg/ml,) approaches thepractical limit of solubility for a cytoxic agent of its potency that isadministered parenterally. Orally administered compounds can be lesssoluble, but they must be soluble enough to be absorbed. There is adesire to design platinum drugs with improved aqueous solubility as wellas improved antitumor activity, particularly in cisplatin resistanttumors.

Citation of the above documents is not intended as an admission that anyof the foregoing is pertinent prior art. All statements as to the dateor representation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments. Further, all documents referred to throughout thisapplication are incorporated in their entirety by reference herein.

DISCLOSURE OF THE INVENTION

The invention is directed to the platinum(IV) complex,ammine(chloro)trihydroxo(2-methylpyridine)platinum(IV), having theformula

(hereinafter Compound A), and the pharmaceutically acceptable saltsthereof, to pharmaceutical compositions thereof, and to methods to treattumors using this compound.

Thus, a further aspect of the invention is Compound A for use inmedicine and in particular for use in the treatment of cancer. A stillfurther aspect of the invention relates to use of Compound A in themanufacture of a medicament for the treatment of cancer. Alternatively,there is provided a method of treating cancer comprising administeringto a patient in need thereof, a pharmaceutically effective amount ofCompound A, optionally along with additional medicaments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows structures of compounds A-G and of two additional Ptcomplexes.

MODES OF CARRYING OUT THE INVENTION

Aqueous solubility and antitumor activity, particularly against platinumresistant tumors are important objectives in the development of newplatinum antitumor agents. Compound A has significantly improved aqueoussolubility compared to the currently approved platinum drugs andcompared to cis-amminedichloro(2-methylpyridine)platinum(II) and hasgreater or equivalent antitumor activity to cisplatin and tocis-amminedichloro(2-methylpyridine)platinum(II), particularly inresistant tumors. The latter finding is particularly unexpected ascompounds B, C, D and E (FIG. 1) showed poorer activity. Platinum(IV)compounds in general are considered more inert than platinum(II)compounds and generally show lower antitumor activities than theirplatinum(II) analogs.

In in vivo studies in which the platinum compounds are administered attheir maximum tolerated dose to CH1 tumor containing xenografts, a 32.8days growth delay was achieved with Compound A, equivalent to the growthdelay of 34 days achieved with cisplatin. This is in contrast to thegrowth delay of 0.6 days, achieved with compound E (FIG. 1).

Compound A also exhibited significantly improved aqueous solubilitycompared to cisplatin, carboplatin,cis-amminedichloro(2-methylpyridine)platinum(II) and also some similarplatinum(IV) compounds such as compound B, C, E, F and G (FIG. 1). Thegreater aqueous solubility of Compound A allows for an easierformulation and parental administration of the drug particularly theadministration of the drug by intravenous infusion. A higher aqueoussolubility would allow for a higher drug dose to be intravenouslyadministered in a smaller volume of saline.

The combination of improved aqueous solubility and good antitumoractivity makes Compound A an significant improvement over the currentplatinum drugs and over cis-amminedichloro(2methylpyridine)platinum(II)and sets it apart from other platinum(IV) compounds.

Compound A may be prepared by methods known in the art. Generalpreparation methods for platinum complexes with mixed ammine/alkylamineligands are given by Braddock, P. D.; et al., Chem.-Biol. Interactions1975, 11, 145-161; and Giandomenico, C. M.; et al., Inorg. Chem. 1995,34, 1015-1021.

One synthetic method is to react [PtCl₃(NH₃)]⁻ with 2-methylpyridine toproduce cis-amminedichloro(2-methylpyridine)platinum(II). [PtCl₃(NH₃)]⁻can be prepared from [PtCl₄]²⁻ using methods known in the art(Giandomenico, Inorg. Chem. 1995, supra, and Abrams, J. J., et al.,Inorg. Chim. Acta 1987 131, 3-4). Compound A can be generated from tocis-amminedichloro(2-methylpyridine)platinum(II) by reacting withhydrogen peroxide to produceamminedichlorodihydroxo(2-methylpyridine)platinum(IV), followed byreaction with hydroxide to produce Compound A. Another synthetic methodis to prepare cis-amminedichloro(2-methylpyridine)platinum(II) from[PtCl₄]²⁻ as starting material followed by reaction with hydrogenperoxide, followed by hydroxide to generate Compound A.

Compound A, including its salts, may be administered in the form ofpharmaceutical compositions formulated according to well-knownprinciples. Such compositions may be in the form of solutions ofsuspensions for injection, or to be in capsule, tablet, dragee, or othersolid composition or as a solution or suspension for oral administrationor formulated into pessaries or suppositories, or sustained release formof any of the above. Suitable diluents, carriers, excipients and othercomponents are known. It may be desirable also to formulate acomposition for topical administration such as an ointment or cream, orto be administered as a transdermal patch. Standard and well-knownmethods to formulate compounds similar to Compound A and compounds ofthis type in general are found in Remington's Pharmaceutical Sciences,latest edition, Mack Publishing Co., Easton, Pa.

The pharmaceutical compositions according to the invention may containdosages determined in accordance with conventional pharmaceuticalmethods, suitably to provide active compounds in the dosage range inhumans of from 0.1 to 100 mg/Kg body weight per day, in a single unitdose or in a number of smaller unit doses. Preferred dosage ranges are 1to 30 mg/Kg body weight per day.

The dosage suitable for any particular subject is conveniently optimizedby the ordinary practitioner. Individual dosages depend on the conditionof the subject, the judgment of the practitioner, the severity of thecondition, the mode of administration, and the manner of formulation.Optimization of individual dosages is routine and such individualdosages may fall outside the preferred ranges set forth above.

Compound A may be administered alone or in combination with anotherchemotherapeutic agents such as gemcitabine, etoposide or taxol, eitheras a single treatment or course of treatment or as part of combinedtherapy with other pharmaceuticals to overcome or diminish side effectsor to improve bioavailability, or in combination with other therapiessuch as radiation treatment.

The following examples are for illustration, and are not intended to belimiting of the present invention.

EXAMPLE 1 Synthesis of Compound A

A. Synthesis of K[PtCl₃(2-picoline)]:

K₂[PtCl₄] was ground into a very fine powder with a mortar and pestle.3.5047 g (8.443 mmoles) of K₂[PtCl₄] was placed in a 25 ml round bottomflask and 6-7 ml of dry NMP was added. 0.8648 g (9.286 mmoles) of2-picoline was placed in 3-4 ml of NMP and divided in 5 equal portions.The first portion of 2-picoline was added to the Pt mixture. The mixturewas completely immersed in a 60° C. oil bath and stirred at 1200 rpm.Subsequent portions of 2-picoline was added at 30-35 minutes internals.The rate of 2-picoline addition was 20% every 30-35 minutes. After theaddition of the last portion, the reaction was allowed to proceed foranother 50 to 60 minutes. The reaction solution was orange in color atthe end of the reaction. The reaction solution was allowed to cool toambient temperature. 100 ml of methylene chloride was added to thereaction at ambient temperature. The addition of methylene chloridecaused the precipitation of K[PtCl₃(2-picoline)] and KCl. Theprecipitate was collected by vacuum filtration using a glass frit andwashed with methylene chloride (3×5 ml), followed diethyl ether (3×5ml). The precipitate was dried under vacuum at ambient temperature for16-24 hours and weighed. Yield: 3.8440 g (86.8%). Anal. to Calcd (found)for C₆H₇N₁Cl₃KPt.1.2 K₁Cl₁: C, 13.74 (13.54); H, 1.35 (1.39); N, 2.67(2.59); Cl, 28.51 (28.32). ¹H NMR (300 MHz, DMF-d⁶): 9.12 (d, 1 pyridineH); 7.90 (t, 1 pyridine H); 7.61 (d, 1 pyridine H); 7.40 (t, 1 pyridineH); 3.40 (s, 3 methyl H). ¹⁹⁵Pt NMR (300 MHz, DMF-d⁶): conforms toreference. HPLC retention time: conforms to reference.

B. Synthesis of cis-[PtCl₂(NH₃)(2-picoline)]:

6.819 g (12.50 mmoles) of K[PtCl₃(2-picoline)]. 1.5 KCl was placed in a25 ml round bottom flask and 10 ml of 2.5 N KCl solution added. 8.2688 g(63.12 mmoles) of ammonium acetate trihydrate was dissolved in 25 ml of2.5 N ammonium hydroxide solution and added to the stirring Pt mixture.The total volume of the reaction was ˜35 ml. The orange colored mixturewas immersed in a 45° C. oil bath and was stirred for 1 hour in the darkat >1000 rpm. The orange mixture gradually turned into a yellow coloredmixture. The yellow precipitate was collected by vacuum filtration usinga glass frit and washed with water (2×5 ml) and acetone (3×5 ml). Theprecipitate was dried under vacuum at ambient temperature for 16-24hours and weighed. Yield: 3.8996 g (83%). Anal. Calcd (found) forC₆H₁₀N₂Cl₂Pt: C, 19.16 (19.25); H, 2.68 (2.72); N, 7.45 (7.43); Cl,18.85 ¹H NMR (300 MHz, DMF-d⁶): 9.19 (d, 1 pyridine H); 8.03 (t, 1pyridine H); 7.15 (d, 1 pyridine H); 7.51 (t, 1 pyridine H); 4.39 (bs, 3NH₃ H); 3.34 (s, 3 methyl H). ¹⁹⁵Pt NMR (300 MHz, DMF-d⁶): conforms toreference. HPLC retention time: conforms to reference.

C. Synthesis of c,t,c-[PtCl₂(OH)₂(NH₃)(2-picoline)]:

5.0 ml of water and 5.0 ml 30% H₂O₂ was added to a suspension of 3.142 gof ZD0473 in 15-20 ml of heptane. This mixture was stirred and heated to˜80° C. for 2 hours. The mixture was cooled to room temperature and thenstirred for 1 hour in an ice bath. The bright yellow solid was collectedby vacuum filtration and washed with water and methanol. The product wasdried under vacuum at ambient temperature overnight. Yield: 2.975 g(87%). Anal. Calcd (found) for C₆H₁₂N₂Cl₂O₂Pt: C, 17.57 (17.67); H, 2.95(2.93); N 63 (6.79) Cl, 17.29 (17.39).

D. Synthesis of PtCl(OH)₃(NH₃)(2-picoline)] (Compound A):

0.246 g of LiOH.H₂O was dissolved in 5 ml of water. 2.402 g ofc,t,c-[PtCl₂(OH)₂(NH₃) (2-picoline)] from paragraph C was suspended inthis solution. The mixture was stirred overnight at ambient temperature.The yellow solid gradually dissolved overnight. The pH of the solutionwas adjusted to 7. The solvent was removed under reduced pressure toyield a yellow solid. In order to wash away the resulting LiCl, thesolid was stirred in 10 ml of ethanol for 30 min. The mixture wascentrifuged and the supernatant decanted off. This washing process wasrepeated until lithium chloride was removed. The product was dried undervacuum at ambient temperature overnight. Yield: 1.209 (50%). Anal. Calcd(found) for C₆H₁₃N₂ClO₃Pt.2H₂O.0.12 LiCl: C, 16.65 (16.45); H, 3.96(4.04); N, (6.47 (6.75); Cl, 9.17 (9.47).

E. Alternative Synthesis of [PtCl(OH)₃NH₃)(2-picoline)] (Compound A):

3.83 g (0.0098 mol) of c,t,c-[PtCl₂(OH)₂(NH₃)(2-picoline)] fromparagraph C was dissolved in 55.8 ml of H₂O (approx. 10 minutes tocompletely dissolve). LiOH.H₂O (1eq, 0.401 g , 0.0098 mol) was added tothis solution. The solution was stirred for 18 hours at room temperaturewhile protected from light. After 18 hours, the pH of the solution(which may be basic, eg ˜pH 9) was adjusted to ˜pH 7 using a smallamount of 1M HCl. The solvent was removed on a roto-evaporator to give ayellow solid that was transferred to a centrifuge tube. Ethanol (20 ml)was added and the suspension was shaken vigorously for 10 minutes beforecentrifuging the solid until it settled to the bottom of the tube. Theliquid was decanted and washing procedure was repeated. The solid wascollected and dried under vacuum.

The solid [PtCl(OH)₃(NH₃)(2-picoline)] (2.7 g, 0.0069 mol) was gentlyheated in 6 ml of H₂O until it had fully dissolved (it was not necessaryto heat to the boiling point). Once fully dissolved, the solution wasallowed to stand at room temperature for 3 hours then cooled to ˜4° C.and allowed to stand overnight. (If crystals do not form, inducecrystallization by seeding, scratching or agitating.) The crystals werecollected by filtration and dried under vacuum.

Yield of [PtCl(OH)₃(NH₃)(2-picoline)] was 2.4 g, 0.0061 mol. Anal.Calcd. (found) for C₆H₁₃N₂ClO₃Pt: C:18.40(17.98); H:3.34(3.31);N:7.15(6.88); Cl:9.05(9.11)O:12.25 (12.37).

EXAMPLE 2 Solubility of Compound A

The aqueous solubility of Compound A and some other platinum complexesat ambient conditions is shown in Table 1.

TABLE 1 Ratio to Ratio to Solubility of Ratio to Solubility ofSolubility cis-ammine(2- bis-butyrato-ammine- Aqueous of methylpyridine)(cyclohexylamine) Compounds Solubility cisplatin Dichloroplatinum (II)dichloro platinum (IV) cisplatin 1 mg/ml 1 1.4 5 carboplatin 14 mg/ml 1420 70 cis-ammine(2-methylpyridine) 0.7 mg/ml 0.7 1 3.5dichloroplatinum(II) Compound G (FIG. 1) 0.2 mg/ml 0.2 0.3 1 Compound F(FIG. 1) <1 mg/ml <1 <1.4 <5 Compound A (FIG. 1) 80 mg/ml 80 114.3 400Compound B (FIG. 1) ˜5 mg/ml 5 7.1 25 Compound C (FIG. 1) 20 mg/ml 2028.6 100 Compound D (FIG. 1) 80 mg/ml 80 114.3 400 Compound E (FIG. 1)15 mg/ml 15 21.4 75

As shown in Table 1, the solubility of Compound A is 80-fold that ofcisplatin and significantly higher than the solubility of the other twolisted prior art compounds. The only comparative compound solubilitysimilar to that of Compound A is that of Compound D.

EXAMPLE 3 Anti Tumor Activity

Compounds were tested against human cancer cell lines grown in cellculture according to established procedures (described by Holford, etal., British J. Cancer 1998 77(3) 366-373). The results are shown belowin Table 2. The results are given in concentrations (μM) necessary toachieve a 50% decrease in cell proliferation (IC₅₀), with resistancefactors in brackets for the cisplatin-resistant cell lines. The celllines bred to be resistant to cisplatin have the designation R, as in41MR.

TABLE 2 Compounds 41M 41MR CH1 CH1R A2780 2780R cisplatin 0.26   1.20.11    0.71 0.33   5.2 (4.6) (6.5) (15.7)  carboplatin 3.3   8.8 1.3  61.8   25.9 (2.7) (4.5) (14.4)  cis-ammine(2-methylpyridine)- 5.6  6 2.2  2.1 2.5 12 dichloro platinum (II) (1.1) (1)   (4.8) Compound F(FIG. 1) 0.5   0.6 1.0   0.4 0.4    1.84 (1.2) (4)   (4.6) Compound G(FIG. 1) 0.072    0.048 0.0066    0.024 NA NA  (0.67) (3.7) Compound A(FIG. 1) 25 45 6 12 9.5 41 (1.8) (2)   (4.3) Compound B (FIG. 1) 10 193.4   7.4 4.5 36 (1.9) (2.2) (8)   Compound C (FIG. 1) 27 26 8.6 17 8.645  (0.96) (1.8) (5.2) Compound D (FIG. 1) >100 >100    46 88 47 >100   (1.9) Compound E (FIG. 1) 78 78 18 46 10.5 62 (1)   (2.6) (5.9)

As seen, Compound A has a resistance ratio comparable to that of knownanti-tumor drugs. Of particular interest, however, is that itsresistance ratio and IC₅₀ are dramatically better than those of CompoundD, the only compound compared to Compound A which has similar solubilitycharacteristics.

EXAMPLE 4 Combined Solubility, Activity Comparisons

To determine the Pt(IV) compounds with the optimal aqueous solubilityand antitumor activity, the Pt(TV) compounds (Compounds A-E) werecompared with the Pt(II) analog,cis-amminedichloro(2-methylpyridine)platinum(II). For each of the Pt(IV)compounds, the ratio of Pt(IV) compound solubility over the Pt(II)compound solubility were calculated (Sol_(pt(IV))/Sol_(pt(II))). Foreach of the Pt(IV) compounds, the ratio of IC₅₀ of the Pt(IV) compoundover the IC₅₀ of Pt(II) compound in various cell lines were calculated(IC_(50-Pt(IV))/IC_(50-Pt(II))). The mean ratio of(Sol_(Pt(IV))/Sol_(Pt(II))) over (IC_(50-Pt(IV))/IC_(50-Pt(II))) wasthen calculated for each compound. The larger the ratio of(Sol_(Pt(IV))/Sol_(Pt(II))) over (IC_(50-Pt(IV))/IC_(50-Pt(II))), themore optimal the compound. Compound A was found the have the mostoptimal aqueous solubility and antitumor activity of the Pt(IV)compounds listed.

Compound Compound Compound Compound Compound A B C D E(Sol_(Pt(IV))/Sol_(Pt(II))) 114.3 7.1 28.6 114.3 21.4(IC_(50-Pt(IV))/IC_(50-Pt(II))) 3.8 1.8 3.4 18.8 4.2 A2780 cell line(IC_(50-Pt(IV))/IC_(50-Pt(II))) 3.4 3.0 3.8 8.3 5.2 A2780R cell line(IC_(50-Pt(IV))/IC_(50-Pt(II))) 2.7 1.5 3.9 20.9 8.2 CH1 cell line(IC_(50-Pt(IV))/IC_(50-Pt(II))) 5.7 3.5 8.1 41.9 21.9 CH1R cell line(IC_(50-Pt(IV))/IC_(50-Pt(II))) 4.5 1.8 4.8 17.9 13.9 41M cell line(IC_(50-Pt(IV))/IC_(50-Pt(II))) 7.5 3.2 4.3 16.7 13 41MR cell line meanratio of (Sol_(Pt(IV))/Sol_(Pt(II))) over 27.7 3.2 6.5 6.9 2.7(IC_(50-Pt(IV))/IC_(50-Pt(II)))

One having ordinary skill in the art to which this invention pertains inreviewing the specification and claims which follow would appreciatethat there are equivalents to those claimed aspects of the invention.The inventors intend to encompass those equivalents within thereasonable scope of the claimed invention.

We claim:
 1. A pharmaceutical composition comprising the compoundammine(chloro)trihydroxo(2-methylpyridine)platinum(IV), of the formula

or a pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier or diluent.
 2. A method to treat ovarian cancer in asubject comprising administering to a subject in need thereof apharmaceutically effective amount of the compoundammine(chloro)trihydroxo(2-methylpyridine)platinum(IV), of the formula

or a pharmaceutically acceptable salt thereof.